This invention relates to a novel gene that activates Fibroblast Growth Factor Receptor 2 (FGFR2).
The fibroblast growth factors (FGFs) are a family of related proteins with roles in mitogenesis, differentiation, wound healing, and organogenesis (Basilico et al. Adv. Cancer Res. 59:115-165, 1992). The biological responses of FGFs are mediated through specific high-affinity receptor tyrosine kinases (Givol and Yayon, FASEB J. 6:3362-3369, 1992). Four distinct classes of fibroblast growth factor receptor (FGFR) that encode structurally related proteins have been identified: FGFR1/Flg, FGFR2/Bek, FGFR3, and FGFR4 (Dionne et al., EMBO J. 9:2685-2692, 1990); Miki et al., Science 251:72-75, 1991; Keegan et al., Proc. Natl. Acad. Sci. USA 88:1095-1099, 1991; and Partanen et al., EMBO J. 10: 1347-1354, 1992).
Activating mutations (i.e. mutations, leading to highly phosphorylated receptors, that are associated with various cancer states) in transmembrane domains of EGF receptor and FGFR3 were reported in rat leukemia and human achondroplasia, respectively (Ben-Levy et al., J. Biol. Chem. 267:17304-17313, 1992; and Shiang et al., Cell, 78:335-342, 1994). Furthermore, several growth factor receptors have been found to be activated by chromosomal rearrangement in cancer cells (Sawyers et al., Cell 77:171-173, 1994), including EGFR in leukemias, NGFR (TRK) in colon and thyroid carcinomas, HGFR (MET) in gastric carcinomas, RET in thyroid carcinomas, ALK in lymphomas, and PDGFRxcex2 in myeloid leukemias.
One chromosomal rearrangement associated with chronic myelomonocytic leukemia was shown to create an expressed fusion between a novel gene sequence, tel, and the tyrosine kinase domain of the growth factor receptor PDGFRxcex2 (Golub et al., Cell 77:307-316, 1994). tel appears to be a member of the ets gene family, members of which encode transcription factors. Genes other than growth factor receptors (GFRs), are activated and cause tumorigenesis by gene fusions resulting from chromosomal arrangement. For example, a majority of the cases of chronic myeloma leukemia (CML) contain a chromosomal rearrangement between chromosome 9 and 22 (t(9;22)(q34;q11) that results in a fusion of the oncogene xcex1-abl, a cytoplasmic kinase, to bcr, which promotes myeloid tumorigenesis and is used as a diagnostic for the malignancy (Bernards et al., Mol. Cell Biol. 7:3231-3236, 1987; and Konopka et al., Proc. Nat""l. Acad. Sci. 82:1810-1813, 1985).
We have cloned and sequenced rat and human FRAG1 cDNAs (SEQ ID NO: 5 and 11, respectively). Chromosomal rearrangements resulting in a fusion of FRAG1 to fibroblast growth factor receptor 1 (FGFR2) causes a potent activation of FGFR2. Based on these discoveries, the present invention provides compositions and methods related to the isolated rat and human FRAG1 genes.
Accordingly, one embodiment of the invention is an isolated nucleic acid that comprises: (a) a sequence of at least 15 contiguous nucleotides of a native FRAG1 nucleic acid or the complement thereof; or (b) a sequence of at least 100 nucleotides having at least 70%, more preferably at least 80%, and yet more preferably at least 90%, and most preferably at least 95% nucleotide sequence similarity with a native FRAG1 nucleic acid (or a complement thereof), particularly with the rat or human FRAG1 nucleic acid (SEQ ID NO: 5 and 11, respectively). According to another embodiment, the isolated nucleic acid encodes a polypeptide sequence having only silent or conservative substitutions to a native rat or human FRAG 1 polypeptide. According to another embodiment, the isolated nucleic acid encodes a native or wild-type rat or human FRAG1 polypeptide (SEQ ID NO: 6 and 12, respectively). According to another embodiment, the isolated nucleic acid encodes a polypeptide that, when expressed as an in-frame fusion with FGFR2, stimulates the transforming activity and autophosphorylation of FGFR2.
Another embodiment of the invention is a cell that comprises such FRAG1 nucleic acids, including expression vectors that are suitable for expression of recombinant FRAG1 polypeptides in a host cell. Such cells can be used to making FRAG1 polypeptides by culturing the cells under conditions suitable for expression of the FRAG1 polypeptide, followed by isolation of the expressed FRAG1 polypeptide from the cell by conventional methods.
FRAG1 nucleic acids are useful for detecting an abnormality in a chromosome (e.g., a chromosomal rearrangement such as a fusion of FRAG1 to another gene that, when expressed, produces a fusion polypeptide) of a subject comprising the steps of: incubating chromosomes of a subject that comprises a chromosomal abnormality (e.g., a rearrangement resulting in a fusion of FRAG1 to another gene) with a FRAG1 nucleic acid probe or primer under conditions that cause the probe or primer to hybridize specifically with a native FRAG1 sequence. Hybridization of the probe or primer to the subject""s chromosomes is compared with hybridization to a normal control chromosome, i.e., a chromosome known to lack the abnormality, thereby allowing the abnormality to be detected. Detection of chromosomal abnormalities can be accomplished, for example, by fluorescence in situ hybridization or nucleic acid amplification techniques. Such chromosome abnormalities may be diagnostic for disease states such as a neoplasia (e.g., an osteosarcoma).
Another embodiment of the invention is isolated FRAG1 polypeptides, e.g., polypeptides encoded by a FRAG1 nucleic acid as described above. For example, FRAG1 polypeptides according to various embodiments of the invention include polypeptides that comprise at least 10 consecutive amino acids of a native rat or human FRAG1 polypeptide; polypeptides having at least 70% amino acid sequence homology to a native rat or human FRAG1 polypeptide; and full-length native FRAG1 polypeptides.
Another embodiment of the invention is an antibody that is specific for a native FRAG1 polypeptide.
FRAG1 genes can be isolated from species other than rat or human by contacting nucleic acids of the species with a FRAG1 probe or primer under at least moderately stringent nucleic acid hybridization conditions and isolating the FRAG1 gene to which the probe or primer hybridizes. For example, a cDNA or genomic library of the species can be screened with a FRAG1 probe, or primer, or mRNA or genomic DNA can be subjected to a nucleic acid amplification procedure to amplify a FRAG1 homolog of the species. In an alternative method of obtaining a FRAG1 gene of a species other than rat or human, an expression library comprising a plurality of cells that each express a recombinant polypeptide is contacted with a FRAG1-specific antibody under conditions that cause the FRAG1-specific antibody to specifically bind to a recombinant polypeptide encoded by a FRAG1 gene, thereby identifying a cell that expresses the FRAG1 gene. The FRAG1 gene can then be isolated from the cell that expresses the FRAG1 gene.
The foregoing and other aspects of the invention will become more apparent from the following detailed description and accompanying drawings.